1. Field of Invention
The invention generally relates to the preparation of blood films or smears on microscope slides or similar substrates. While applicable to all techniques for evaluation of the resulting smears, the method especially is adapted for use in connection with automated blood cell analysis using computerized pattern recognition systems, commonly known as automated differential analyzers.
2. Description of Prior Art
Monolayers of cells have been prepared by a number of techniques, for example, by wedging, i.e., "push smears", by coverslip techniques and more recently by spinning. The major advantages of the spinning technique are the uniform coverage of at least a major portion of a 1".times.3" microscope slide area and the good cell morphology analysis which can be achieved. Especially for automated differential analyzers, these advantages have made spun smears very attractive. The amount of whole blood required for these spun smears is in the range of 100 to 200 microliters per slide, depending on the density of cells per quantity of sample liquid considered adequate, the use of whole blood or diluted blood and also on the amount of blood wasted, for example, blood remaining on the wall of test tubes, in dispensers and in the dilutors. If less fluid is dispensed, only an irregular, star-shaped pattern is obtained, which covers only part of the slide area.
The most frequent use of these blood films is for differential white cell counts and for assessment of the red cell morphology. In normal blood samples there are about 600 erythrocytes (red cells) for every leukocyte (white cell). Hence, in order to find a minimum of 100 leukocytes for a differential count, enough of a slide surface has to be scanned to cover an average of 6.times.10.sup.4 erythrocytes. To find these white cells in a reasonable time, of the order of 2 minutes total, one would ideally like to have a blood film with closely spaced, but not touching or overlapping cells, with well defined platelets, few damaged cells, uniform and reproducible cell size for assessment of degrees of microcytosis and macrocytosis, well developed pallor of normal red cells and freedom from artifacts and debris.
Assuming a slide area of 1".times.3", normal blood and a distribution, where on the average two red cells are contained in an area of 16 microns by 16 microns, a volume of blood which would contain enough cells to form the above described monolayer can be calculated easily. About 1.5 microliters of whole blood are required for this. If, on the other hand, a blood film containing 1,000 white cells from normal blood is desired, an area of only 76.8 mm.sup.2 would be needed, constituting, for example, a circle of less than 1 cm diameter on the slide. The whole blood volume required for this would be only about 1.5/25=0.06 microliters. Even if the whole slide is covered, only about 1% of the blood used becomes part of the monolayer, while about 99% is spun off, i.e. 150 microliters versus 1.5 microliters.
If the proper slide preparation method can be found, it is apparent from the above described wastage that spun slides could be prepared with a substantially smaller amount of blood. The amount of blood sample required to prepare a film becomes important especially in the following applications: fingersticks; pediatric sticks; need to prepare multiple slides from the same donor; research applications, using blood from small animals for example; and application of spun layers outside hematology with small sample requirements.